The disclosed invention is generally in the field of detection and amplification of nucleic acids, and in particular involves detection and amplification based on target-specific ligation of oligonucleotides.
Numerous techniques for detection and/or amplification of nucleic acids are known. A number of these involve ligation of oligonucleotides. Such techniques include ligase chain reaction (LCR; Wiedmann et al., PCR Methods Appl. 3(4):S51-64 (1994); U.S. Pat. No. 5,516,663 to Backman et al.), ligation-mediated polymerase chain reaction (LMPCR; Rodriguez and Akman, Electrophoresis 19:646-652 (1998)), ligation-dependent polymerase chain reaction (LD-PCR; Park et al., Am J Pathol 149(5):1485-1491 (1996)), oligonucleotide ligation assay (OLA; Tobe et al., Nucleic Acids Res. 24:3728-3732 (1996)), and ligation of padlock probes or open circle probes (Nilsson et al., Science 265:2085-2088 (1994); U.S. Patent No. 5, 854,033 to Lizardi).
These techniques generally involve ligation of DNA ends by T4 DNA ligase or other DNA ligases. Many ligation-based techniques involve ligation of the oligonucleotides hybridized to another nucleic acid strand and generally depend on the ends of the oligonucleotides being adjacent to each other. Some DNA ligases can ligate the ends of DNA strands hybridized to an RNA strand. T4 DNA ligase is an example of this (Hsuih et al., Quantitative detection of HCV RNA using novel ligation-dependent polymerase chain reaction, American Association for the Study of Liver Diseases, Abstract 1002 (Chicago, Ill., Nov. 3-7, 1995)).
T4 RNA ligase joins a 3xe2x80x2-hydroxyl-terminated acceptor oligoribonucleotide to a 5xe2x80x2-phosphate-terminated donor oligoribonucleotide. (Silber et al., Proc. Natl. Acad. Sci. USA 69:3009 (1972)). As its name denotes, T4 RNA ligase is principally know for ligation of RNA ends. Studies with T4 RNA ligase revealed that it could catalyze the formation of a phosphodiester bond between the 3xe2x80x2 hydroxyl and 5xe2x80x2 phosphate of short oligonucleotides (Brennan et al., Methods in Enzymology 100(Part B):38-52 (1983); Tessier et al., Analytical Biochem. 158:171-178 (1986)). The reactions required high concentrations of nucleic acids and were not efficient. These studies did not attempt ligation of DNA strands when hybridized to RNA. RNA-directed RNA ligase has been used to ligate RNA probes hybridized to a target sequence (U.S. Pat. No. 5,807,674). This ligation was used to detect specific sequences to which pairs of RNA probes would hybridize. T4 DNA ligase was the preferred ligase.
RNA molecules hybridized to RNA or DNA strands have been ligated. For example, ligation of RNA padlock probes hybridized to mRNA was described by Brian Johnston (IBCs Fifth Annual International Conference on xe2x80x9cAntisense: DNA and RNA Based Therapeutics,xe2x80x9d Feb. 2-3, 1998, Coronado, Calif.). Moore and Sharpe, Science 256:922 (1992), describe circularization of RNA using a DNA xe2x80x9csplint.xe2x80x9d
It would be useful to improve the efficiency of RNA detection and amplification techniques involving ligation.
It is therefore an object of the present invention to provide techniques to allow ligation-mediated detection of RNA sequences.
It is a further object of the present invention to provide techniques to allow ligation-mediated amplification of RNA sequences.
It is a further object of the present invention to provide techniques to allow ligation-dependent detection of RNA sequences.
It is a further object of the present invention to provide techniques to allow ligation-dependent amplification of RNA sequences.
It is a further object of the present invention to provide techniques to allow ligation-mediated or ligation-dependent amplification and detection of RNA sequences.
It is a further object of the present invention to provide techniques to allow ligation-mediated or ligation-dependent amplification and detection of RNA sequences for the purpose of quantitative analysis of RNA expression levels.
Disclosed are techniques for detection of nucleic acids, amplification of nucleic acids, or both, involving ligation by T4 RNA ligase of DNA strands hybridized to an RNA strand. These techniques are particularly useful for the detection of RNA sequences and for amplification of nucleic acids from, or dependent on, RNA sequences. Many known ligation-based detection and amplification techniques are improved through the use of T4 RNA ligase acting on DNA strands or ends. Such techniques include ligase chain reaction (LCR), ligation combined with reverse transcription polymerase chain reaction (RT PCR), ligation-mediated polymerase chain reaction (LMPCR), polymerase chain reaction/ligation detection reaction (PCR/LDR), ligation-dependent polymerase chain reaction (LD-PCR), oligonucleotide ligation assay (OLA), ligation-during-amplification (LDA), ligation of padlock probes, open circle probes, and other circularizable probes, and iterative gap ligation (IGL).
A preferred technique is the target-mediated ligation of open circle probes (see U.S. Pat. No. 5, 854,033 to Lizardi; also known as padlock probes; see Nilsson et al., Science 265:2085-2088 (1994)) where the target nucleic acid is RNA. This can be followed by rolling circle amplification (RCA) of the ligated open circle probe resulting in target-dependent nucleic acid amplification. Exponential rolling circle amplification (ERCA) can provide even more amplification.
It has been discovered that T4 RNA ligase can efficiently ligate DNA ends of nucleic acid strands hybridized to an RNA strand. In particular, this ligation is more efficient than the same ligation carried out with T4 DNA ligase. Thus, techniques involving ligation of DNA ends of nucleic acid strands hybridized to RNA can be performed more efficiently by using T4 RNA ligase.